Determining the Antibacterial Effect of Mentha Longifolia Essential Oil on Cariogenic Bacteria and Its Compounds: an in vitro Study

Statement of the Problem: Continuous use of chemical agents to reduce the number of cariogenic bacteria leads to adverse effects; therefore, in recent years, many studies have focused on plant-based substances. Purpose: This study explores the antibacterial effects of Mentha Longifolia (M. longifolia) essential oil on Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), and Lactobacillus as cariogenic microorganisms and determines the compounds in it. Materials and Method: In this experimental study, S. mutans, S. sobrinus, and Lactobacillus isolates were collected from the saliva samples of five children with severe early childhood caries (S-ECC). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of M. longifolia essential oil were determined by Broth microdilution method. Chlorhexidine 0.2% and phosphate-buffered normal saline (pH= 7.0) were used as positive and negative controls, respectively. The chemical composition of M. longifolia essential oil was evaluated by gas chromatography-mass spectrometry. The data were analyzed with a paired t-test and the p below 0.05 was considered significant. Results: The MIC and MBC ratios for S. mutans were 3.12% and 6.25%, for S. sobrinus were 6.25% and 12.5%, and Lactobacillus were 3.12% and 6.25%, respectively. Chemical analysis of M. longifolia essential oil showed that 34 various compounds. Piperitone oxide (27.59%), Transcariophylline (14.55%), and 2-cyclohexane-1-one (12.24%) were the major constituents. Conclusion: M. longifolia essential oil has both growth inhibitory and bactericidal effects on all the three species of bacteria. This antibacterial effect was similar against S. mutans and Lactobacillus, which was greater than S. sobrinus, thus, it can be used as a supplementary for caries prevention compounds.

Reduction in the number of oral microorganisms prevents dental caries [3]. One of the most popular antibacterial chemical agents used in the mouth is chlorhexidine. The antimicrobial effect of chlorhexidine is due to its cationic property, which gives it persistent bactericidal and bacteriostatic effect on tooth surfaces. However, loss of taste sensation, teeth discoloration, burning sensation of the oral mucosa, and dry mouth has been reported as side effects of chlorhexidine [4][5]. Other popular antimicrobial agents, antibiotics, and antimicrobial mouthwashes have numerous side effects despite their usefulness [5][6][7], so the search for new antimicrobial agents with minimal side effects is warranted. Northern, and Northeast Iran. It spreads to the East and some other places of Iran [8]. Due to its many biological properties, it has been studied extensively. Its medicinal properties include anti-inflammatory, anti-mutagenic, antioxidant, anti-rheumatic, antispasmodic, anti-viral, anti-candida, anti-platelet adhesion, muscle relaxant, and Cyclooxygenase inhibitor properties [9][10][11][12]. Some studies have evaluated the antimicrobial activity of the essential oil of this plant [12][13]. Hydrophobicity is one of the most important features of essential oils and accounts for their antimicrobial effect. This property enables the essential oils to penetrate the membrane lipids and mitochondria of bacteria, making the membrane more permeable and causing the release of ions and other intracellular contents of bacteria [12].
Studies have shown antibacterial properties of M. longifolia against many gram-positive and gramnegative bacteria [12][13]. In addition, several researches were done to evaluate the components of M. longifolia essential oil that reported different major compositions of the plant [14][15] and the composition depended on climatic or geographic conditions [16] .
The aim of this study was to investigate the antibacterial effect of M. longifolia essential oil on oral cariogenic bacteria, such as S. mutans, S. sobrinus, and Lactobacillus. In addition to saliva sampling of children with severe early childhood caries (S-ECC) [17], stand-ard strains were also used and the results were compared with chlorhexidine. In addition, components of essential oil were also investigated.

Materials and Method
This experimental study has been approved by Babol For essential oil extraction, nine hundred and eighty grams of fresh M. longifolia aerial parts was chopped and the essential oil extracted through distillation and Clevenger method (Schottduran-Germany).

Preparation of bacterial strains
S. mutans, S. sobrinus, and Lactobacillus strains were collected from the non-duplicative saliva samples of five children [18][19] with S-ECC. Consent was obtained orally after justifying the dimensions of the research.
Children in the study had no medical problems as well as not taking antibiotics, steroids, and topical fluoride therapy history in a month before sampling. In brief, 2 ml of unstimulated saliva was taken from each child.
The children spotted the saliva, as instructed in 3 to 5minutes in sterile plastic containers with screws [20].  [21] was added to all wells.

Detection of bacteria using specific tests
Then a medium containing 100 bacteria was added to each well. Well number 12 in each row contained 0.2% chlorhexidine, which served as a positive control. After inoculation of all the wells, the microplate was placed on a shaker for 30 seconds to achieve a uniform mixture. The mixture was incubated at 35°C for 24 hours in an anaerobic jar. The first well in which no growth was observed was designated as the MIC. The results were repeated three times.

Determination of minimum bactericidal concentration (MBC)
Ten microliter was removed from MIC dilution and a few higher dilutions and cultured on blood agar and MRS agar media. A concentration of essential oil in which no bacterial growth was observed on the medium was reported as MBC. The results were repeated three times.

Gas chromatography analysis of essential oil
Helium gas (with a purity of 99.999%) was injected into the column of a gas chromatography machine at a rate of 0.8 ml/min. The temperature of the column was raised from 40°C to 208°C at a rate of 5°C/min. and spectral mass data of the NIST9 and wiley7 database [22].

Data analysis
Data from the study were analyzed using the statistical software SPSS version 24.00. Paired Sample T-Test was performed for each group. p less than 0.05 was considered significant (p≤ 0.05).

MIC and MBC test results for M. longifolia essential oil
According to Table 1

Statistical analysis
The results of a comparative study of the mean MIC and MBC in the group of M. longifolia and chlorhexidine essential oils are given in Table 2. Results of the t-test of the two dependent samples demonstrated significance level less than 0.05 in all groups. This is interpreted that a significant difference between M. longifolia and chlorhexidine exist in all groups.

Identification of components
The gas chromatogram obtained from this M. longifolia essential oil can be seen in Figure 1.  (Table 2).

Discussion
Plant-derived antimicrobial compounds kill bacteria by mechanisms different from antibiotics, and this difference is clinically important in the treatment of infections caused by resistant microbial strains [23].
M. longifolia has a wide range of antimicrobial activities against various bacteria, yeasts, insects, and other organisms. Previous study has reported that M. longifolia essential oil has more antimicrobial activity than hydro alcoholic extract [9]. In 2016, Raeisi et al. [24], evaluated the effects of Mentha Piperita (M. Piperita) and NaCl on S. mutans, and found that M. Piperita hydro alcoholic extract had no antibacterial properties   Table 3 against S. mutans in agar and in disk diffusion methods,  for Actinomycosis. The differences between this study and the present study could be due to the extract and the type of strains used. In addition, the concentration range of the extracts was 0.18-100µg/ml, while in the present study; the concentration range of the essential oil was 0.50µg/ml. In 2016, Ghasemi et al. [28] found that the Akhbar et al. [32], the main components of M. longifolia essential oil were decarvone and limonene. In another study, pulegone has been reported as the main constituent of the essential oil [33], but pulegone was not found in the essential oil of M. longifolia in the current study. The major polyphenol in methanolic leaf extracts of M. longifolia was rosmarinic acid, based on the study done by Elansary HO. et al. [34].
The composition of essential oil of one plant species may differ from that of the same species due to different regional conditions, which may be related to differences in the harvest season, times spent in essential oil extraction, differences in geographical areas, and even differences in the parts of the plant [16,35]. Bakkali et al. [36] found that compounds such as limonene, linalool, di-limonene, gamma-terpinene, p-cymene, alpha- From the results of the present study, it can be said that M. longifolia has an antimicrobial effect on oral microbes. Since chlorhexidine mouthwash can cause discoloration and microbial imbalance [41], and amoxicillin, which is an antibiotic of choice in dentistry, can cause bacterial resistance [42], there is a need to introduce a substance that does not have these characteristics. The efficiency of M. longifolia essential oil against cariogenic microorganisms was confirmed in this study.
However, a clinical study is recommended due to variable factors in the oral environment and the difference between the oral environment and the laboratory enviroment [43].

Conclusion
The results of the present study showed that the MIC for S. mutans was 3.12%, S. sobrinus was 6.25%, and Lactobacillus was 3.12%. The highest composition of M.
longifolia essential oil is Piperitone oxide, Trans-Caryophyllene, 2-cyclohexane-1-one. Results from this study showed that M. longifolia essential oil had a growth inhibitory and lethal effect on all three cariogenic microorganisms, although this effect was less than chlorhexidine.